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Spinally administered dynorphin A produces long-lasting allodynia: involvement of NMDA but not opioid receptors
Pain 72 (1997) 253–260
Spinally administered dynorphin A produces long-lasting allodynia: involvement of NMDA but not opioid receptors Tinna M. Laughlin a, Todd W. Vanderah b, Jason Lashbrook b, Mike L. Nichols b, Mike Ossipov b, Frank Porreca b, George L. Wilcox a , c ,* a
Department of Pharmacology, 3–249 Millard Hall, University of Minnesota Medical School, Minneapolis, MN 55455, USA b Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA c Program in Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA Received 16 January 1997; revised version received 18 April 1997; accepted 1 May 1997
Abstract The endogenous opioid peptide dynorphin A has non-opioid effects that can damage the spinal cord when given in high doses. Dynorphin has been shown to increase the receptive field size of spinal cord neurons and facilitate C-fiber-evoked reflexes. Furthermore, endogenous dynorphin levels increase following damage to the spinal cord, injury to peripheral nerves, or inflammation. In this study, sensory processing was characterized following a single, intrathecal injection of dynorphin A (1-17) in mice. A single intrathecal injection of dynorphin A (1-17) (3 nmol, i.t.) induced mechanical allodynia (hind paw, von Frey filaments) lasting 70 days, tactile allodynia (paint brush applied to flank) lasting 14 days, and cold allodynia (acetone applied to the dorsal hind paw) lasting 7 days. Similarly, dynorphin A (2-17) (3 nmol, i.t.), a non-opioid peptide, induced cold and tactile allodynia analogous to that induced by dynorphin A (1-17), indicating the importance of non-opioid receptors. Pretreatment with the NMDA antagonists, MK-801 and LY235959, but not the opioid antagonist, naloxone, blocked the induction of allodynia. Post-treatment with MK-801 only transiently blocked the dynorphin-induced allodynia, suggesting the NMDA receptors may be involved in the maintenance of allodynia as well as its induction. We have induced a long-lasting state of allodynia and hyperalgesia by a single intrathecal injection of dynorphin A (1-17) in mice. The allodynia induced by dynorphin required NMDA receptors rather than opioid receptors. This result is consistent with results in rats and with signs of clinically observed neuropathic pain. This effect of exogenously administered dynorphin raises the possibility that increased levels of endogenous dynorphins associated with spinal cord injuries may participate in the genesis and maintenance of neuropathic pain. 1997 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Dynorphin A; Allodynia; NMDA; Spinal cord
1. Introduction Dynorphin A (1-17), a proposed endogenous ligand for the kappa opioid receptor, interacts with the NMDA receptor to produce non-opioid effects, such as decreased blood flow and paralysis (Shukla and Lemaire, 1994). Dynorphin is unique among endogenous opioid peptides in producing hind limb paralysis, and this paralysis is blocked by NMDA but not opioid receptor antagonists (Caudle and Isaac, 1988b; Bakshi and Faden, 1990). Because this dynorphin-
* Corresponding author. Tel.: +1 612 6251474; fax: +1 612 6258408; e-mail: [email protected]
induced paralysis is accompanied by neuronal cell loss in the spinal cord (Caudle and Isaac, 1987; Long et al., 1988; Bakshi et al., 1992), it has been suggested that increased dynorphin peptide levels lead to excitotoxicity through activation of NMDA receptors (Dubner and Ruda, 1992). Several lines of evidence indicate that dynorphin may have a pathological role in addition to the accepted role of opioid peptides in analgesia (Dubner and Ruda, 1992). First, dynorphin is located in intrinsic neurons in lamina I, II, and V of the spinal cord dorsal horn (Ruda et al., 1988; Miller and Seybold, 1989); these locations overlap with those of neurons responding to noxious inputs. Second, dynorphin levels increase after spinal cord injury (Cox et al., 1985), during peripheral inflammation (Iadarola et al., 1988) and in
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an animal model of neuropathic pain (Kajander et al., 1990). During spinal cord injury, dynorphin peptide levels accumulate at the site of injury in proportion to the severity of injury (Faden et al., 1985). Administration of dynorphin antiserum diminished the severity of spinal cord injury (Faden, 1990), suggesting that the endogenous dynorphin peptide may have a role in the pathology of spinal cord injury. Third, intrathecal administration of dynorphin produces hind limb flaccidity (Faden and Jacobs, 1984; Stevens and Yaksh, 1986; Caudle and Isaac, 1987). Finally exogenously administered dynorphin enhances spinal cord synaptic transmission by facilitating C-fiber-evoked responses (Knox and Dickenson, 1987; Caudle and Isaac, 1988a), and increasing receptive field size to mechanical stimulation in a naloxone-insensititive manner (Hylden et al., 1991). The increase in dynorphin levels after a noxious insult, the ability of dynorphin to induce motor dysfunction, and dynorphin’s modulation of synaptic transmission all suggest that dynorphin may play a role in the pathology of neuropathic pain states. Previously, we have shown that a single intrathecal injection of dynorphin produces a long-term allodynic state in rats (Vanderah et al., 1996). The purpose of this study was to examine the ability of non-paralytic doses of dynorphin A (1-17) to increase the sensitivity to sensory stimulation in mice. We found that a single intrathecal injection of dynorphin A (1-17) induced a long-lasting allodynia in mice, which was blocked by NMDA receptor antagonists but was insensitive to opioid receptor antagonism. Thus the allodynia induced by dynorphin is mediated by NMDA receptors rather than opioid receptors.
2. Materials and methods 2.1. Animals Male ICR mice (Harlan) weighing 17–27 g were used in all experiments. Mice were maintained in cages with free access to food and water and kept on a 12-h light-dark schedule in the University of Minnesota Research Animal Resources facilities. These experiments were ap-proved by the Institutional Animal Care and Use Committee. 2.2. Drugs Dynorphin A (1-17), used in the tactile and cold allodynia experiments, and naloxone were from Sigma Chemical Co. (St. Louis, MO). Dynorphin A (1-17), used in the von Frey filament experiments, was a gift from NIDA. Dynorphin A (2-17) was a generous gift from Professor Victor Hruby (Tucson, AZ). LY235959 was a gift from Lilly Research Laboratories (Indianapolis, IN). MK-801 was from Merck Chemical Co. (Fort Washington, PA). All drugs were diluted in 0.9% saline and administered in a volume of 5 ml by intrathecal (i.t.) injections to awake animals as previously described (Hylden and Wilcox, 1980).
2.3. Nociceptive behavioral tests Tactile allodynia was determined by stroking the mouse’s flank with a paint brush as previously described (Yaksh and Harty, 1988). The responses were ranked: 0 = no response, or an orientation change; 1 = mild/transient squeaking, or mild avoidance of brush; 2 = sustained/loud squeaking, aggressive avoidance, or biting the brush. Cold allodynia was determined by a modification of the method previously described (Choi et al., 1994). The response to a 10-ml drop of acetone from PE10 tubing applied to each hind paw was ranked: 0 = no response; 1 = foot lifted and/or light shake; 2 = hard/long shake, or squeaking. Mechanical sensitivity was determined with von Frey filaments. We constructed a modified set of von Frey filaments as previously described (Willenbring and Stevens, 1996). Briefly, the filament was glued into the end of a 30-cm plastic rod, such that the filament paralleled the rod. Using the modified filaments we were able to stimulate the dorsal side of the hind paw and avoid lifting the mouse’s foot, which may occur with application of the standard set of von Frey filaments to the plantar side of the mouse’s foot. In this study we examined the frequency of response to two filaments; the innocuous 2.44 filament (0.4 mN) was used to assess mechanical allodynia, and the presumably noxious 4.08 filament (5.1 mN) was used to assess mechanical hyperalgesia. We were unable to unambiguously determine the fiber types activated by these filaments in mice. We therefore estimated the nociceptive threshold of the mouse hind paw dorsum (below 5.1 mN) and then used the withdrawal response to the 5.1-mN filament to observe hyperalgesia. Admittedly, this filament exerts less force than that used by others for determining hyperalgesia on the plantar surface of the rat hind paw (Carlton and Hargett, 1995); however, we believe that the species and site of stimulation differences, together with the withdrawal responses we observed, justify this assertion. The filament was applied to the point of bending three times to the dorsal surface of the left and right hind paw for a total of six applications per mouse for each filament. For each time point the percent response frequency of foot withdrawals was expressed as (number of positive responses)/6 × 100. Mice were placed in individual 2 liter beakers with bedding on the bottom for all behavior tests and allowed to adjust to the surroundings for about 30 min before behavioral testing. All behavioral tests were administered prior to treatment, during the first hour after treatment, and then daily for up to 3 weeks. Due to the subjective nature of these methods, some of the experiments were repeated with the observer being blinded to the drugs administered, and similar results were obtained in the blinded experiments. 2.4. Statistical analysis All animals receiving intrathecal treatments were included in the analysis; no non-responding outliers were
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removed. Individual tactile and cold allodynia scores were converted to a percent maximum possible score (% MPS), so that 100% represents a score of 2. A mean and standard error of the mean (SEM) was calculated for each group from the % MPS. Time course data were converted to area under the curve (AUC) by summing individual scores and dividing by the number time points summed; then mean ± SEM were generated from these values. The data were tested for significance using analysis of variance (ANOVA), and statistical differences between groups were further analyzed with Dunnett’s test for multiple comparisons to a control (saline group).
3. Results A single intrathecal injection of 3 nmol dynorphin A (117) produced both tactile and cold allodynia in mice, whereas an intrathecal injection of saline had no effect during the 14 days (Fig. 1). Ten minutes after injection, dynor-
Fig. 2. Dose-response curves of dynorphin-induced allodynia. A: Tactile allodynia. B: Cold allodynia. Both 3 and 10 nmol, but not 1 nmol, dynorphin induced allodynia significantly greater than saline treatment. The mean ± SEM represents the AUC generated from an individual’s % MPS for days 0–14 after injection; n = 8–16 mice per group.
Fig. 1. Time course of the effect of i.t. treatment with 3 nmol dynorphin A (1-17) (closed circles) compared to saline (open squares). A: Tactile allodynia, determined by stroking the mouse’s flank with a paint brush and ranking the response as described in the Materials and methods section, lasted for 14 days after injection. B: Cold allodynia, determined by applications of acetone to each hind paw and ranking the response as described in the Materials and methods section, lasted for 7 days after injection. The post-injection times are abbreviated as ‘m’ for min and ‘d’ for days. The mean ± SEM represents the percent maximum possible score (% MPS); n = 8–16 mice per group.
phin induced a 53% maximum possible score (% MPS) for tactile allodynia and 38% MPS for cold allodynia. Dynorphin-induced tactile allodynia remained significantly greater than the saline-treated mice for 14 days, while dynorphin-induced cold allodynia was detectable for only 7 days. To ensure that the repeated stimulation applied during the first hour after dynorphin injection was not required for manifestation of the allodynia, we tested tactile and cold allodynia only on day 3. Mice that were tested only on day 3 after injection of 3 nmol dynorphin A (1-17) showed a tactile (56 ± 15% MPS) and cold (31 ± 9.2% MPS) allodynia similar to that of mice receiving repetitive stimulation; thus, repetitive stimulation did not sensitize the mice to the test stimulus. Dynorphin A (1-17) induced both tactile and cold allodynia in a dose-related manner (Fig. 2). Ten nmol dynorphin A (1-17) induced not only tactile and cold allodynia similar to that produced by 3 nmol dynorphin A (1-17) but also temporary hind limb flaccidity that lasted approximately 5 min. Due to the partial paralysis induced by 10 nmol dynorphin A (1-17), subsequent studies pharmacologically characterized the allodynia using the 3-nmol dose. Because dynorphin A (1-17) is an endogenous opioid peptide, we next examined the ability of the universal opioid
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produce significant allodynia (Fig. 4). Thirty-minute pretreatment with 3.6 pmol LY235959, a competitive NMDA receptor antagonist, blocked dynorphin-induced tactile and cold allodynia (Fig. 4). However, 3.6 pmol LY235959 followed by a saline injection induced both tactile and cold allodynia by itself for 14 days. Nevertheless, pretreatment with 36 fmol LY235959, a dose which was not allodynic alone, reduced dynorphin-induced tactile and cold allodynia (Fig. 4). MK801, administered 30 min or 1 day after 3 nmol dynorphin A (1-17), temporarily alleviated both tactile and cold allodynia (Fig. 5). Thus pretreatment with MK801 completely blocked dynorphin allodynia, while MK801 post-treatment produced only transient relief of allodynia. These results imply that the NMDA receptor is required for both induction and maintenance of dynorphin-induced allodynia. In order to determine if dynorphin altered the mechanical sensitivity, mechanical allodynia and hyperalgesia were
Fig. 3. Lack of participation of opioid receptors in dynorphin-induced allodynia. A:Tactile allodynia. B: Cold allodynia. Twenty-minute pretreatment with 0.3 pmol naloxone did not inhibit tactile allodynia induced by 3 nmol dynorphin. Naloxone (0.3 pmol) followed by saline had no effect. Three nmol dynorphin A (2-17), the non-opioid peptide, induced a similar tactile allodynia to that induced by the opioid peptide, dynorphin A (1-17). The mean ± SEM represents the AUC generated from an individual’s % MPS for days 0–7 after injection. *P , 0.05 from saline treatment; n = 7–16 mice per group.
antagonist, naloxone, to block the induction of allodynia. A 20-min pretreatment with naloxone (0.3 pmol i.t.), a dose effective in antagonizing morphine-induced analgesia in the hot-water tail flick assay (data not shown), did not inhibit dynorphin A (1-17)-induced tactile and cold allodynia (Fig. 3). This dose of naloxone followed by a saline injection did not produce any significant allodynia (Fig. 3). In addition, the non-opioid peptide dynorphin A (2-17) produced tactile allodynia for 14 days and cold allodynia for 7 days, effects similar to those produced by the opioid peptide, dynorphin A (1-17) (Fig. 3). Together, these two results suggest that the dynorphin-induced allodynia is not mediated by opioid receptors. Dynorphin has been suggested to interact with NMDA receptors (Massardier and Hunt, 1989; Chen et al., 1995); therefore, we tested the ability of NMDA receptor antagonists to block dynorphin-induced allodynia. Three and 9 nmol of MK-801, a non-competitive NMDA receptor antagonist, administered i.t. 30 min before dynorphin A (1-17) blocked the dynorphin-induced tactile and cold allodynia (Fig. 4). MK801 followed by a saline injection did not
Fig. 4. Participation of NMDA receptors in dynorphin-induced allodynia. A: Tactile allodynia. B: Cold allodynia. The box on the left shows results with dynorphin and saline alone; these data are also presented in Fig. 3. The middle box shows results from a 30-min pretreatment with MK801, and the right box shows results from pretreatment with LY235959. Dynorphin pretreatment with either 3 or 9 nmol MK-801 did not differ from the saline group. Dynorphin pretreatment with 3.6 pmol LY235959 did not differ from the saline group. Five-minute pretreatment with 36 fmol LY235959, a non-allodynic dose, partially reduced the allodynia induced by 3 nmol dynorphin. The mean ± SEM represents the AUC generated from an individual’s % MPS for days 0–7 after injection. *P , 0.05 from saline treatment; P , 0.05 from dynorphin group; n = 6–16 mice per group.
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Fig. 5. Effect of pre- or post-treatment with MK801 on dynorphin-induced tactile allodynia. Individual symbols at the top of the graph indicate when MK801 was administered; dynorphin was administered at time point zero for all groups. The time course of 3 nmol dynorphin alone is represented by the solid circles, and saline alone is represented by the solid diamonds (both also shown in Fig. 1). MK801 (9 nmol, i.t.), administered 30 min (open triangles) after 3 nmol dynorphin, blocked allodynia at 60 min after the dynorphin injection; subsequently, on day 1, the allodynia returned. Similarly, MK801 (9 nmol, i.t.), administered 1 day (open diamonds) after dynorphin, relieved the allodynia induced by dynorphin on that day only; subsequently, on day 2 and afterwards, the allodynia returned. Nine nmol MK801 given 30 min prior (open squares) to 3 nmol dynorphin, blocked the appearance of allodynia at least through day 2 and was not different from saline until day 7 induction of allodynia (also shown in Fig. 6). The mean ± SEM represents the % MPS; n = 8–16 mice per group.
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ment of the NMDA receptors in the induction of the allodynia. Post-treatment with MK801 transiently blocked dynorphin-induced allodynia indicating the involvement of NMDA receptors in the maintenance of the allodynic state. Although dynorphin is the proposed endogenous kappa opioid receptor ligand, it has also been suggested to interact with the NMDA receptor. Dynorphin augmented [3H]glutamate binding to NMDA receptors and blocked the increase of [3H]MK801 binding induced by glutamate and glycine (Massardier and Hunt, 1989). Patch-clamp analysis of isolated trigeminal neurons revealed that dynorphin attenuated NMDA receptor-mediated currents by shortening the mean open time and decreasing the probability of channel opening, apparently through interaction with the redox-modulatory site (Chen et al., 1995). Similarly, dynorphin-induced paralysis was reduced by NMDA receptor antagonists but not by opioid receptor antagonists (Caudle and Isaac, 1987; Long et al., 1988; Bakshi et al., 1992). In agreement with the literature, our studies have demonstrated that dynorphininduced allodynia is blocked by NMDA receptor but not
determined with von Frey filaments. A single intrathecal injection of 3 nmol dynorphin A (1-17) increased the sensitivity for at least 70 days to both the 0.4- and 5.1-mN von Frey filaments (Fig. 6). We attribute this prolonged allodynia with the von Frey filaments to a higher sensitivity of this measurement compared with the flank brushing and hind paw acetone tests. The response frequency to the 0.4-mN filament increased from 0 to 33% by 60 min after dynorphin injection (Fig. 6A). The response frequency to the 5.1-mN filament increased from 56 to 83% by 60 min after dynorphin injection and reached a maximum response of 91% on day 3 after injection (Fig. 6B). Thus, dynorphin induced a long-lasting, dose-dependent (Fig. 7) mechanical allodynia and hyperalgesia, in a manner similar to that observed in the tactile and cold allodynia tests.
4. Discussion The present study demonstrates that a single intrathecal injection of dynorphin A (1-17) is capable of producing a long-lasting allodynic state in mice. Dynorphin A (1-17) induced long-lasting tactile allodynia, cold allodynia, mechanical allodynia, and mechanical hyperalgesia. This effect seems to be mediated by non-opioid receptors, notably NMDA receptors. Dynorphin A (2-17), the non-opioid analog of dynorphin A (1-17), induced a similar long-lasting allodynic state. LY235959 and MK801, but not naloxone, blocked the dynorphin allodynia, indicating the involve-
Fig. 6. Time course of mechanical sensitivity after i.t. treatment with 3 nmol dynorphin A (1-17) (solid circles) compared to saline (open squares). A: Mechanical allodynia measured with the 0.4-mN von Frey filament lasted for at least 70 days after dynorphin injection. B: Mechanical hyperalgesia measured with the 5.1-mN von Frey filament also lasted for 70 days after dynorphin injection. The mean ± SEM represents the percent response frequency of foot withdrawals to six stimulations; n = 9 mice per group.
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Fig. 7. Dose-response curves of mechanical sensitivity following i.t. administration of dynorphin A (1-17). Solid circles represent the 0.4-mN filament dose-response curve, and solid squares represent the 5.1-mN filament dose-response curve. The dashed line represents the baseline response for each filament. The mean ± SEM represents the AUC generated from an individual’s response frequency percentage for days 0–14 after injection; n = 8–16 mice per group.
opioid receptor antagonists in both mouse and rat. Thus, dynorphin-induced injuries involve NMDA rather then kappa opioid receptors. Many studies have demonstrated the involvement of NMDA receptors in nociceptive transmission, neuropathic pain and synaptic plasticity (for review, see Haley and Wilcox, 1992). NMDA receptor antagonists blocked the decrease in mechanical threshold (Ren and Dubner, 1993), the increase in receptive field size and the hyperalgesia following inflammation (Ren et al., 1992). NMDA receptor antagonists blocked signs of hyperalgesia and allodynia in rat models of neuropathic pain (Seltzer et al., 1991; Mao et al., 1992). NMDA receptor activation contributes to spinal cord neuronal plasticity as observed in nociceptive thermal hyperalgesia (Aanonsen and Wilcox, 1987), studies of wind-up (Davies and Lodge, 1987; Dickenson and Sullivan, 1987), and central sensitization (Woolf and Thompson, 1991; Ma and Woolf, 1995). It is reasonable to speculate that the same NMDA receptor-dependent mechanisms involved in wind-up and central sensitization are involved in dynorphin’s induction of long-lasting allodynia and hyperalgesia. Dynorphin-induced allodynia may involve three mechanisms, all of which may involve dynorphin-induced excitotoxicity and central sensitization. First, dynorphin-induced allodynia may result from ischemic injury following a dynorphin-induced decrease in blood flow (Long et al., 1987; Thornhill et al., 1989). In vitro application of dynorphin to cultured spinal cord neurons did not induce cell death as seen in vivo (Long et al., 1994), suggesting that dynorphin-induced reduction in blood flow is the mechanism of its neurotoxicity. In addition, coadministration with the vasodialator, hydralazine, inhibited dynorphin-induced paralysis (Long et al., 1994). Transient ischemia itself has been shown to induce allodynia in rats (Hao et al., 1991). Ischemia entails increased levels of lactic acid and free
radicals, an increased release of excitatory amino acids and fatty acids, and breakdown of the blood-brain barrier, all which could lead to cell damage and death. Second, dynorphin-induced allodynia may be due to a directly mediated excitotoxicity. Dynorphin increased the release of excitatory amino acids (Faden, 1992; Skilling et al., 1992), an action which could lead to excitotoxicity. Dynorphin-induced paralysis is associated with increased levels of nicotinamide adenine dinucleotide phosphate (NADPH)-diphorase, a marker for nitric oxide-producing cells, suggesting that dynorphin can increase levels of nitric oxide in the spinal cord (Hu et al., 1996). Recently, dynorphin was shown to potentiate NMDA-induced currents at low glycine concentrations, but to inhibit NMDA-induced currents at high glycine concentrations (Brauneis et al., 1996). This potentiation was reduced by kynurenic acid, the strychnine-insensitive glycine receptor antagonist; thus it was postulated that dynorphin acts as an agonist at the glycine site to potentiate NMDA currents. In addition, pretreatment with 1-aminocyclopropanecarboxylic acid, a partial agonist at the strychnine-insensitive glycine receptor, inhibited the paralysis and necrosis induced by dynorphin (Long and Skolnick, 1994). A third possible mechanism of dynorphin-induced allodynia may involve disinhibition, which could result in excessive excitation. That dynorphin decreased both [3H]MK801 binding (Massardier and Hunt, 1989) and NMDA-induced currents (Chen et al., 1995) suggests an antagonist action of dynorphin on NMDA receptors. Such a direct antagonistic action of dynorphin on the NMDA receptor would require a disinhibitory mechanism to produce an increased excitatory state. Dynorphin may decrease activity of a population of tonically active GABAergic or glycinergic interneurons by blocking NMDA receptors located on these inhibitory interneurons. In support of this proposal, extracellular and intracellular recordings of substantia gelatinosa neurons in the rat spinal cord show that application of the GABA antagonist, bicuculline, increased neuronal input resistance and responses to dorsal root stimulation, suggesting that tonic GABAergic inhibition on substantia gelatinosa neurons is substantial (Magnuson and Dickenson, 1991). In addition, blockade of glycine receptors with strychnine has been shown to produce allodynia (Yaksh et al., 1986; Sherman and Loomis, 1994), an action blocked by NMDA receptor antagonists (Yaksh, 1989). Furthermore, both strychnine and bicuculline increase the hyperalgesia in a rat model of neuropathic pain (Yamamoto and Yaksh, 1993). Dynorphin also decreased dorsal root potentials, which are thought to reflect presynaptic GABA-mediated inhibition of primary afferent fibers (Stewart and Isaac, 1991). Randic and colleagues suggested that dynorphin interacts with receptors on inhibitory interneurons to induce excitation indirectly (Randic et al., 1995). Thus, dynorphin-induced inhibition of a tonically active inhibitory system would be expected to produce a state of increased excitation. Such a disinhibitory mechan-
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ism could also explain the allodynia produced by the competitive NMDA antagonist, LY235959. In conclusion, we have shown that a single injection of dynorphin A (1-17) produces a long-lasting allodynia and hyperalgesia in mice, states consistent with the signs of clinically observed neuropathic pain. This action required NMDA receptors rather than opioid receptors. These results suggest the possibility that the increased levels of endogenous dynorphin associated with many sustained injuries may participate in excitotoxic changes which could initiate neuropathic pain. Thus, dynorphin may have both a physiological (through opioid receptors) and a pathological (through NMDA receptors) role in acute and chronic pain states. In the future, dynorphin-induced allodynia may find use as a pharmacological, non-surgical model to study the mechanisms of neuropathic pain.
Acknowledgements We thank Kelley F. Kitto for outstanding technical support. LY235959 was a generous gift from the Eli Lilly Company, dynorphin A (1-17) was a generous gift from NIDA, and dynorphin A (2-17) was a generous gift from Dr. Victor Hruby. This study was supported by NIDA/K02-DA-00145 and NIDA/R01-DA-04274 to G.L.W.; NIDA training grant T32 DA07097 supported T.M.L. This study was also supported in part by DA 06284 and DA 04248; F.P. is the recipient of an RSDA (KO2 DA00185) from NIDA.
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Spinally administered dynorphin A produces long-lasting allodynia: involvement of NMDA but not opioid receptors Tinna M. Laughlin a, Todd W. Vanderah b, Jason Lashbrook b, Mike L. Nichols b, Mike Ossipov b, Frank Porreca b, George L. Wilcox a , c ,* a
Department of Pharmacology, 3–249 Millard Hall, University of Minnesota Medical School, Minneapolis, MN 55455, USA b Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA c Program in Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA Received 16 January 1997; revised version received 18 April 1997; accepted 1 May 1997
Abstract The endogenous opioid peptide dynorphin A has non-opioid effects that can damage the spinal cord when given in high doses. Dynorphin has been shown to increase the receptive field size of spinal cord neurons and facilitate C-fiber-evoked reflexes. Furthermore, endogenous dynorphin levels increase following damage to the spinal cord, injury to peripheral nerves, or inflammation. In this study, sensory processing was characterized following a single, intrathecal injection of dynorphin A (1-17) in mice. A single intrathecal injection of dynorphin A (1-17) (3 nmol, i.t.) induced mechanical allodynia (hind paw, von Frey filaments) lasting 70 days, tactile allodynia (paint brush applied to flank) lasting 14 days, and cold allodynia (acetone applied to the dorsal hind paw) lasting 7 days. Similarly, dynorphin A (2-17) (3 nmol, i.t.), a non-opioid peptide, induced cold and tactile allodynia analogous to that induced by dynorphin A (1-17), indicating the importance of non-opioid receptors. Pretreatment with the NMDA antagonists, MK-801 and LY235959, but not the opioid antagonist, naloxone, blocked the induction of allodynia. Post-treatment with MK-801 only transiently blocked the dynorphin-induced allodynia, suggesting the NMDA receptors may be involved in the maintenance of allodynia as well as its induction. We have induced a long-lasting state of allodynia and hyperalgesia by a single intrathecal injection of dynorphin A (1-17) in mice. The allodynia induced by dynorphin required NMDA receptors rather than opioid receptors. This result is consistent with results in rats and with signs of clinically observed neuropathic pain. This effect of exogenously administered dynorphin raises the possibility that increased levels of endogenous dynorphins associated with spinal cord injuries may participate in the genesis and maintenance of neuropathic pain. 1997 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Dynorphin A; Allodynia; NMDA; Spinal cord
1. Introduction Dynorphin A (1-17), a proposed endogenous ligand for the kappa opioid receptor, interacts with the NMDA receptor to produce non-opioid effects, such as decreased blood flow and paralysis (Shukla and Lemaire, 1994). Dynorphin is unique among endogenous opioid peptides in producing hind limb paralysis, and this paralysis is blocked by NMDA but not opioid receptor antagonists (Caudle and Isaac, 1988b; Bakshi and Faden, 1990). Because this dynorphin-
* Corresponding author. Tel.: +1 612 6251474; fax: +1 612 6258408; e-mail: [email protected]
induced paralysis is accompanied by neuronal cell loss in the spinal cord (Caudle and Isaac, 1987; Long et al., 1988; Bakshi et al., 1992), it has been suggested that increased dynorphin peptide levels lead to excitotoxicity through activation of NMDA receptors (Dubner and Ruda, 1992). Several lines of evidence indicate that dynorphin may have a pathological role in addition to the accepted role of opioid peptides in analgesia (Dubner and Ruda, 1992). First, dynorphin is located in intrinsic neurons in lamina I, II, and V of the spinal cord dorsal horn (Ruda et al., 1988; Miller and Seybold, 1989); these locations overlap with those of neurons responding to noxious inputs. Second, dynorphin levels increase after spinal cord injury (Cox et al., 1985), during peripheral inflammation (Iadarola et al., 1988) and in
0304-3959/97/$17.00 1997 International Association for the Study of Pain. Published by Elsevier Science B.V. PII S0304-3959 (97 )0 0046-8
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an animal model of neuropathic pain (Kajander et al., 1990). During spinal cord injury, dynorphin peptide levels accumulate at the site of injury in proportion to the severity of injury (Faden et al., 1985). Administration of dynorphin antiserum diminished the severity of spinal cord injury (Faden, 1990), suggesting that the endogenous dynorphin peptide may have a role in the pathology of spinal cord injury. Third, intrathecal administration of dynorphin produces hind limb flaccidity (Faden and Jacobs, 1984; Stevens and Yaksh, 1986; Caudle and Isaac, 1987). Finally exogenously administered dynorphin enhances spinal cord synaptic transmission by facilitating C-fiber-evoked responses (Knox and Dickenson, 1987; Caudle and Isaac, 1988a), and increasing receptive field size to mechanical stimulation in a naloxone-insensititive manner (Hylden et al., 1991). The increase in dynorphin levels after a noxious insult, the ability of dynorphin to induce motor dysfunction, and dynorphin’s modulation of synaptic transmission all suggest that dynorphin may play a role in the pathology of neuropathic pain states. Previously, we have shown that a single intrathecal injection of dynorphin produces a long-term allodynic state in rats (Vanderah et al., 1996). The purpose of this study was to examine the ability of non-paralytic doses of dynorphin A (1-17) to increase the sensitivity to sensory stimulation in mice. We found that a single intrathecal injection of dynorphin A (1-17) induced a long-lasting allodynia in mice, which was blocked by NMDA receptor antagonists but was insensitive to opioid receptor antagonism. Thus the allodynia induced by dynorphin is mediated by NMDA receptors rather than opioid receptors.
2. Materials and methods 2.1. Animals Male ICR mice (Harlan) weighing 17–27 g were used in all experiments. Mice were maintained in cages with free access to food and water and kept on a 12-h light-dark schedule in the University of Minnesota Research Animal Resources facilities. These experiments were ap-proved by the Institutional Animal Care and Use Committee. 2.2. Drugs Dynorphin A (1-17), used in the tactile and cold allodynia experiments, and naloxone were from Sigma Chemical Co. (St. Louis, MO). Dynorphin A (1-17), used in the von Frey filament experiments, was a gift from NIDA. Dynorphin A (2-17) was a generous gift from Professor Victor Hruby (Tucson, AZ). LY235959 was a gift from Lilly Research Laboratories (Indianapolis, IN). MK-801 was from Merck Chemical Co. (Fort Washington, PA). All drugs were diluted in 0.9% saline and administered in a volume of 5 ml by intrathecal (i.t.) injections to awake animals as previously described (Hylden and Wilcox, 1980).
2.3. Nociceptive behavioral tests Tactile allodynia was determined by stroking the mouse’s flank with a paint brush as previously described (Yaksh and Harty, 1988). The responses were ranked: 0 = no response, or an orientation change; 1 = mild/transient squeaking, or mild avoidance of brush; 2 = sustained/loud squeaking, aggressive avoidance, or biting the brush. Cold allodynia was determined by a modification of the method previously described (Choi et al., 1994). The response to a 10-ml drop of acetone from PE10 tubing applied to each hind paw was ranked: 0 = no response; 1 = foot lifted and/or light shake; 2 = hard/long shake, or squeaking. Mechanical sensitivity was determined with von Frey filaments. We constructed a modified set of von Frey filaments as previously described (Willenbring and Stevens, 1996). Briefly, the filament was glued into the end of a 30-cm plastic rod, such that the filament paralleled the rod. Using the modified filaments we were able to stimulate the dorsal side of the hind paw and avoid lifting the mouse’s foot, which may occur with application of the standard set of von Frey filaments to the plantar side of the mouse’s foot. In this study we examined the frequency of response to two filaments; the innocuous 2.44 filament (0.4 mN) was used to assess mechanical allodynia, and the presumably noxious 4.08 filament (5.1 mN) was used to assess mechanical hyperalgesia. We were unable to unambiguously determine the fiber types activated by these filaments in mice. We therefore estimated the nociceptive threshold of the mouse hind paw dorsum (below 5.1 mN) and then used the withdrawal response to the 5.1-mN filament to observe hyperalgesia. Admittedly, this filament exerts less force than that used by others for determining hyperalgesia on the plantar surface of the rat hind paw (Carlton and Hargett, 1995); however, we believe that the species and site of stimulation differences, together with the withdrawal responses we observed, justify this assertion. The filament was applied to the point of bending three times to the dorsal surface of the left and right hind paw for a total of six applications per mouse for each filament. For each time point the percent response frequency of foot withdrawals was expressed as (number of positive responses)/6 × 100. Mice were placed in individual 2 liter beakers with bedding on the bottom for all behavior tests and allowed to adjust to the surroundings for about 30 min before behavioral testing. All behavioral tests were administered prior to treatment, during the first hour after treatment, and then daily for up to 3 weeks. Due to the subjective nature of these methods, some of the experiments were repeated with the observer being blinded to the drugs administered, and similar results were obtained in the blinded experiments. 2.4. Statistical analysis All animals receiving intrathecal treatments were included in the analysis; no non-responding outliers were
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removed. Individual tactile and cold allodynia scores were converted to a percent maximum possible score (% MPS), so that 100% represents a score of 2. A mean and standard error of the mean (SEM) was calculated for each group from the % MPS. Time course data were converted to area under the curve (AUC) by summing individual scores and dividing by the number time points summed; then mean ± SEM were generated from these values. The data were tested for significance using analysis of variance (ANOVA), and statistical differences between groups were further analyzed with Dunnett’s test for multiple comparisons to a control (saline group).
3. Results A single intrathecal injection of 3 nmol dynorphin A (117) produced both tactile and cold allodynia in mice, whereas an intrathecal injection of saline had no effect during the 14 days (Fig. 1). Ten minutes after injection, dynor-
Fig. 2. Dose-response curves of dynorphin-induced allodynia. A: Tactile allodynia. B: Cold allodynia. Both 3 and 10 nmol, but not 1 nmol, dynorphin induced allodynia significantly greater than saline treatment. The mean ± SEM represents the AUC generated from an individual’s % MPS for days 0–14 after injection; n = 8–16 mice per group.
Fig. 1. Time course of the effect of i.t. treatment with 3 nmol dynorphin A (1-17) (closed circles) compared to saline (open squares). A: Tactile allodynia, determined by stroking the mouse’s flank with a paint brush and ranking the response as described in the Materials and methods section, lasted for 14 days after injection. B: Cold allodynia, determined by applications of acetone to each hind paw and ranking the response as described in the Materials and methods section, lasted for 7 days after injection. The post-injection times are abbreviated as ‘m’ for min and ‘d’ for days. The mean ± SEM represents the percent maximum possible score (% MPS); n = 8–16 mice per group.
phin induced a 53% maximum possible score (% MPS) for tactile allodynia and 38% MPS for cold allodynia. Dynorphin-induced tactile allodynia remained significantly greater than the saline-treated mice for 14 days, while dynorphin-induced cold allodynia was detectable for only 7 days. To ensure that the repeated stimulation applied during the first hour after dynorphin injection was not required for manifestation of the allodynia, we tested tactile and cold allodynia only on day 3. Mice that were tested only on day 3 after injection of 3 nmol dynorphin A (1-17) showed a tactile (56 ± 15% MPS) and cold (31 ± 9.2% MPS) allodynia similar to that of mice receiving repetitive stimulation; thus, repetitive stimulation did not sensitize the mice to the test stimulus. Dynorphin A (1-17) induced both tactile and cold allodynia in a dose-related manner (Fig. 2). Ten nmol dynorphin A (1-17) induced not only tactile and cold allodynia similar to that produced by 3 nmol dynorphin A (1-17) but also temporary hind limb flaccidity that lasted approximately 5 min. Due to the partial paralysis induced by 10 nmol dynorphin A (1-17), subsequent studies pharmacologically characterized the allodynia using the 3-nmol dose. Because dynorphin A (1-17) is an endogenous opioid peptide, we next examined the ability of the universal opioid
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produce significant allodynia (Fig. 4). Thirty-minute pretreatment with 3.6 pmol LY235959, a competitive NMDA receptor antagonist, blocked dynorphin-induced tactile and cold allodynia (Fig. 4). However, 3.6 pmol LY235959 followed by a saline injection induced both tactile and cold allodynia by itself for 14 days. Nevertheless, pretreatment with 36 fmol LY235959, a dose which was not allodynic alone, reduced dynorphin-induced tactile and cold allodynia (Fig. 4). MK801, administered 30 min or 1 day after 3 nmol dynorphin A (1-17), temporarily alleviated both tactile and cold allodynia (Fig. 5). Thus pretreatment with MK801 completely blocked dynorphin allodynia, while MK801 post-treatment produced only transient relief of allodynia. These results imply that the NMDA receptor is required for both induction and maintenance of dynorphin-induced allodynia. In order to determine if dynorphin altered the mechanical sensitivity, mechanical allodynia and hyperalgesia were
Fig. 3. Lack of participation of opioid receptors in dynorphin-induced allodynia. A:Tactile allodynia. B: Cold allodynia. Twenty-minute pretreatment with 0.3 pmol naloxone did not inhibit tactile allodynia induced by 3 nmol dynorphin. Naloxone (0.3 pmol) followed by saline had no effect. Three nmol dynorphin A (2-17), the non-opioid peptide, induced a similar tactile allodynia to that induced by the opioid peptide, dynorphin A (1-17). The mean ± SEM represents the AUC generated from an individual’s % MPS for days 0–7 after injection. *P , 0.05 from saline treatment; n = 7–16 mice per group.
antagonist, naloxone, to block the induction of allodynia. A 20-min pretreatment with naloxone (0.3 pmol i.t.), a dose effective in antagonizing morphine-induced analgesia in the hot-water tail flick assay (data not shown), did not inhibit dynorphin A (1-17)-induced tactile and cold allodynia (Fig. 3). This dose of naloxone followed by a saline injection did not produce any significant allodynia (Fig. 3). In addition, the non-opioid peptide dynorphin A (2-17) produced tactile allodynia for 14 days and cold allodynia for 7 days, effects similar to those produced by the opioid peptide, dynorphin A (1-17) (Fig. 3). Together, these two results suggest that the dynorphin-induced allodynia is not mediated by opioid receptors. Dynorphin has been suggested to interact with NMDA receptors (Massardier and Hunt, 1989; Chen et al., 1995); therefore, we tested the ability of NMDA receptor antagonists to block dynorphin-induced allodynia. Three and 9 nmol of MK-801, a non-competitive NMDA receptor antagonist, administered i.t. 30 min before dynorphin A (1-17) blocked the dynorphin-induced tactile and cold allodynia (Fig. 4). MK801 followed by a saline injection did not
Fig. 4. Participation of NMDA receptors in dynorphin-induced allodynia. A: Tactile allodynia. B: Cold allodynia. The box on the left shows results with dynorphin and saline alone; these data are also presented in Fig. 3. The middle box shows results from a 30-min pretreatment with MK801, and the right box shows results from pretreatment with LY235959. Dynorphin pretreatment with either 3 or 9 nmol MK-801 did not differ from the saline group. Dynorphin pretreatment with 3.6 pmol LY235959 did not differ from the saline group. Five-minute pretreatment with 36 fmol LY235959, a non-allodynic dose, partially reduced the allodynia induced by 3 nmol dynorphin. The mean ± SEM represents the AUC generated from an individual’s % MPS for days 0–7 after injection. *P , 0.05 from saline treatment; P , 0.05 from dynorphin group; n = 6–16 mice per group.
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Fig. 5. Effect of pre- or post-treatment with MK801 on dynorphin-induced tactile allodynia. Individual symbols at the top of the graph indicate when MK801 was administered; dynorphin was administered at time point zero for all groups. The time course of 3 nmol dynorphin alone is represented by the solid circles, and saline alone is represented by the solid diamonds (both also shown in Fig. 1). MK801 (9 nmol, i.t.), administered 30 min (open triangles) after 3 nmol dynorphin, blocked allodynia at 60 min after the dynorphin injection; subsequently, on day 1, the allodynia returned. Similarly, MK801 (9 nmol, i.t.), administered 1 day (open diamonds) after dynorphin, relieved the allodynia induced by dynorphin on that day only; subsequently, on day 2 and afterwards, the allodynia returned. Nine nmol MK801 given 30 min prior (open squares) to 3 nmol dynorphin, blocked the appearance of allodynia at least through day 2 and was not different from saline until day 7 induction of allodynia (also shown in Fig. 6). The mean ± SEM represents the % MPS; n = 8–16 mice per group.
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ment of the NMDA receptors in the induction of the allodynia. Post-treatment with MK801 transiently blocked dynorphin-induced allodynia indicating the involvement of NMDA receptors in the maintenance of the allodynic state. Although dynorphin is the proposed endogenous kappa opioid receptor ligand, it has also been suggested to interact with the NMDA receptor. Dynorphin augmented [3H]glutamate binding to NMDA receptors and blocked the increase of [3H]MK801 binding induced by glutamate and glycine (Massardier and Hunt, 1989). Patch-clamp analysis of isolated trigeminal neurons revealed that dynorphin attenuated NMDA receptor-mediated currents by shortening the mean open time and decreasing the probability of channel opening, apparently through interaction with the redox-modulatory site (Chen et al., 1995). Similarly, dynorphin-induced paralysis was reduced by NMDA receptor antagonists but not by opioid receptor antagonists (Caudle and Isaac, 1987; Long et al., 1988; Bakshi et al., 1992). In agreement with the literature, our studies have demonstrated that dynorphininduced allodynia is blocked by NMDA receptor but not
determined with von Frey filaments. A single intrathecal injection of 3 nmol dynorphin A (1-17) increased the sensitivity for at least 70 days to both the 0.4- and 5.1-mN von Frey filaments (Fig. 6). We attribute this prolonged allodynia with the von Frey filaments to a higher sensitivity of this measurement compared with the flank brushing and hind paw acetone tests. The response frequency to the 0.4-mN filament increased from 0 to 33% by 60 min after dynorphin injection (Fig. 6A). The response frequency to the 5.1-mN filament increased from 56 to 83% by 60 min after dynorphin injection and reached a maximum response of 91% on day 3 after injection (Fig. 6B). Thus, dynorphin induced a long-lasting, dose-dependent (Fig. 7) mechanical allodynia and hyperalgesia, in a manner similar to that observed in the tactile and cold allodynia tests.
4. Discussion The present study demonstrates that a single intrathecal injection of dynorphin A (1-17) is capable of producing a long-lasting allodynic state in mice. Dynorphin A (1-17) induced long-lasting tactile allodynia, cold allodynia, mechanical allodynia, and mechanical hyperalgesia. This effect seems to be mediated by non-opioid receptors, notably NMDA receptors. Dynorphin A (2-17), the non-opioid analog of dynorphin A (1-17), induced a similar long-lasting allodynic state. LY235959 and MK801, but not naloxone, blocked the dynorphin allodynia, indicating the involve-
Fig. 6. Time course of mechanical sensitivity after i.t. treatment with 3 nmol dynorphin A (1-17) (solid circles) compared to saline (open squares). A: Mechanical allodynia measured with the 0.4-mN von Frey filament lasted for at least 70 days after dynorphin injection. B: Mechanical hyperalgesia measured with the 5.1-mN von Frey filament also lasted for 70 days after dynorphin injection. The mean ± SEM represents the percent response frequency of foot withdrawals to six stimulations; n = 9 mice per group.
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Fig. 7. Dose-response curves of mechanical sensitivity following i.t. administration of dynorphin A (1-17). Solid circles represent the 0.4-mN filament dose-response curve, and solid squares represent the 5.1-mN filament dose-response curve. The dashed line represents the baseline response for each filament. The mean ± SEM represents the AUC generated from an individual’s response frequency percentage for days 0–14 after injection; n = 8–16 mice per group.
opioid receptor antagonists in both mouse and rat. Thus, dynorphin-induced injuries involve NMDA rather then kappa opioid receptors. Many studies have demonstrated the involvement of NMDA receptors in nociceptive transmission, neuropathic pain and synaptic plasticity (for review, see Haley and Wilcox, 1992). NMDA receptor antagonists blocked the decrease in mechanical threshold (Ren and Dubner, 1993), the increase in receptive field size and the hyperalgesia following inflammation (Ren et al., 1992). NMDA receptor antagonists blocked signs of hyperalgesia and allodynia in rat models of neuropathic pain (Seltzer et al., 1991; Mao et al., 1992). NMDA receptor activation contributes to spinal cord neuronal plasticity as observed in nociceptive thermal hyperalgesia (Aanonsen and Wilcox, 1987), studies of wind-up (Davies and Lodge, 1987; Dickenson and Sullivan, 1987), and central sensitization (Woolf and Thompson, 1991; Ma and Woolf, 1995). It is reasonable to speculate that the same NMDA receptor-dependent mechanisms involved in wind-up and central sensitization are involved in dynorphin’s induction of long-lasting allodynia and hyperalgesia. Dynorphin-induced allodynia may involve three mechanisms, all of which may involve dynorphin-induced excitotoxicity and central sensitization. First, dynorphin-induced allodynia may result from ischemic injury following a dynorphin-induced decrease in blood flow (Long et al., 1987; Thornhill et al., 1989). In vitro application of dynorphin to cultured spinal cord neurons did not induce cell death as seen in vivo (Long et al., 1994), suggesting that dynorphin-induced reduction in blood flow is the mechanism of its neurotoxicity. In addition, coadministration with the vasodialator, hydralazine, inhibited dynorphin-induced paralysis (Long et al., 1994). Transient ischemia itself has been shown to induce allodynia in rats (Hao et al., 1991). Ischemia entails increased levels of lactic acid and free
radicals, an increased release of excitatory amino acids and fatty acids, and breakdown of the blood-brain barrier, all which could lead to cell damage and death. Second, dynorphin-induced allodynia may be due to a directly mediated excitotoxicity. Dynorphin increased the release of excitatory amino acids (Faden, 1992; Skilling et al., 1992), an action which could lead to excitotoxicity. Dynorphin-induced paralysis is associated with increased levels of nicotinamide adenine dinucleotide phosphate (NADPH)-diphorase, a marker for nitric oxide-producing cells, suggesting that dynorphin can increase levels of nitric oxide in the spinal cord (Hu et al., 1996). Recently, dynorphin was shown to potentiate NMDA-induced currents at low glycine concentrations, but to inhibit NMDA-induced currents at high glycine concentrations (Brauneis et al., 1996). This potentiation was reduced by kynurenic acid, the strychnine-insensitive glycine receptor antagonist; thus it was postulated that dynorphin acts as an agonist at the glycine site to potentiate NMDA currents. In addition, pretreatment with 1-aminocyclopropanecarboxylic acid, a partial agonist at the strychnine-insensitive glycine receptor, inhibited the paralysis and necrosis induced by dynorphin (Long and Skolnick, 1994). A third possible mechanism of dynorphin-induced allodynia may involve disinhibition, which could result in excessive excitation. That dynorphin decreased both [3H]MK801 binding (Massardier and Hunt, 1989) and NMDA-induced currents (Chen et al., 1995) suggests an antagonist action of dynorphin on NMDA receptors. Such a direct antagonistic action of dynorphin on the NMDA receptor would require a disinhibitory mechanism to produce an increased excitatory state. Dynorphin may decrease activity of a population of tonically active GABAergic or glycinergic interneurons by blocking NMDA receptors located on these inhibitory interneurons. In support of this proposal, extracellular and intracellular recordings of substantia gelatinosa neurons in the rat spinal cord show that application of the GABA antagonist, bicuculline, increased neuronal input resistance and responses to dorsal root stimulation, suggesting that tonic GABAergic inhibition on substantia gelatinosa neurons is substantial (Magnuson and Dickenson, 1991). In addition, blockade of glycine receptors with strychnine has been shown to produce allodynia (Yaksh et al., 1986; Sherman and Loomis, 1994), an action blocked by NMDA receptor antagonists (Yaksh, 1989). Furthermore, both strychnine and bicuculline increase the hyperalgesia in a rat model of neuropathic pain (Yamamoto and Yaksh, 1993). Dynorphin also decreased dorsal root potentials, which are thought to reflect presynaptic GABA-mediated inhibition of primary afferent fibers (Stewart and Isaac, 1991). Randic and colleagues suggested that dynorphin interacts with receptors on inhibitory interneurons to induce excitation indirectly (Randic et al., 1995). Thus, dynorphin-induced inhibition of a tonically active inhibitory system would be expected to produce a state of increased excitation. Such a disinhibitory mechan-
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ism could also explain the allodynia produced by the competitive NMDA antagonist, LY235959. In conclusion, we have shown that a single injection of dynorphin A (1-17) produces a long-lasting allodynia and hyperalgesia in mice, states consistent with the signs of clinically observed neuropathic pain. This action required NMDA receptors rather than opioid receptors. These results suggest the possibility that the increased levels of endogenous dynorphin associated with many sustained injuries may participate in excitotoxic changes which could initiate neuropathic pain. Thus, dynorphin may have both a physiological (through opioid receptors) and a pathological (through NMDA receptors) role in acute and chronic pain states. In the future, dynorphin-induced allodynia may find use as a pharmacological, non-surgical model to study the mechanisms of neuropathic pain.
Acknowledgements We thank Kelley F. Kitto for outstanding technical support. LY235959 was a generous gift from the Eli Lilly Company, dynorphin A (1-17) was a generous gift from NIDA, and dynorphin A (2-17) was a generous gift from Dr. Victor Hruby. This study was supported by NIDA/K02-DA-00145 and NIDA/R01-DA-04274 to G.L.W.; NIDA training grant T32 DA07097 supported T.M.L. This study was also supported in part by DA 06284 and DA 04248; F.P. is the recipient of an RSDA (KO2 DA00185) from NIDA.
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